Mutational activation of KRAS represents the most frequently occurring oncogenic driver across human cancers and is associated with poor prognosis and resistance to treatment. Therapeutic options for patients with KRAS mutations have been limited by the lack of direct KRAS inhibitors in clinical trials ? therefore efforts to date have focused on searching for therapies that exploit the principle of synthetic lethality or inhibit downstream signaling cascades activated by KRAS mutations. Genome-wide functional genomics screens have been used to identify synthetic lethal (SL) vulnerabilities associated with KRAS mutant tumors with the hope of identifying singular targets to treat KRAS mutant tumors. Although these functional genomics efforts have contributed to a rich understanding of the genetic dependencies associated with KRAS mutations, such findings have not yet translated to clinical or pre-clinical therapeutic benefit. With the recent emergence of well-characterized, potent, in vivo active direct KRASG12C inhibitors (ARS-1620), it is now possible to directly inhibit this driver-oncogene pharmacologically in an allele-specific manner. The goal of this proposal is to use the novel KRASG12C inhibitor ARS-1620 in a genome-wide assessment of genetic dependencies that complements existing approaches. Aim 1 proposes to identify genes that are responsible for mediating sensitivity and resistance to KRASG12C inhibition using a genome-wide nuclease-dead Cas9-mediated transcriptional repression (CRISPRi) functional genomics platform. Combination therapies targeting KRASG12C can then be derived from knowledge of factors that increase sensitivity to the KRASG12C inhibitor. Preliminary results from CRISPRi screens have raised additional questions regarding the dynamic intracellular regulation of KRASG12C that warrant further investigation. Aim 2 proposes to assess the interactions between receptor tyrosine kinases (RTKs) and KRASG12C in supporting an aberrantly activated GTP state. Additionally, the possibility of therapeutically targeting KRAS mutant cancers with RTK inhibitors will be assessed. Aim 3 proposes to understand the mechanism by which knockdown of the GTPase activating protein (GAP) NF1 confers resistance to KRASG12C inhibition. This result came as a surprise as the tumor suppressor role of NF1 is thought to be negated by KRAS mutation at codon 12. Completion of this proposal will deepen an understanding of mutant KRAS genetic dependencies complementary to existing approaches, inform the development of KRASG12C-targeted combination therapies, and shed light on a potentially novel function of a clinically relevant tumor suppressor.